6alpha-halo-11alpha-hydroxy steroids of the pregnane series and esters thereof



Patented Oct. 10, 1961 United States Patent Gee This invention relatesto the synthesis of valuable steroids and more particularly has for itsits objects the provision of an improved method for preparing 6whalo-9'(11)-dehydrosteroids, and new intermediates utilizable in saidpreparation.

The final products preparable by the process of this invention can berepresented by the general formula wherein the ll-position is saturatedor double-bonded, X is halogen (preferably fiuoro or chloro), Z ishydrogen or hydroxy, and. Y is hydrogen, hydroxy, or acyloxy (especiallythe acyloxy radical of a hydrocarbon car- PIE wherein the 1,2-positionis saturated or double-bonded and X, Y-and Z are as hereinbeforedefined, is subjected to the action of enzymes of an lla-hydroxylatingmicroorganism, thereby yielding the new intermediates of this inventionof the general formula CHZY wherein the 1,2-p'osition is saturated ordouble-bonded, X and Z are as hereinbefore defined, and Y is hydrogen orhydroxy.

This conversion is accomplished by bringing together the startingsteroid and the enzymes of the microorganism in an aqueous mediumcontaining a source of nitrogenous factors and an assimilable source ofcarbon and energy, in the presence of oxygen, and recovering theIla-hydroxy steroid formed. Among the suitable microor ganisms may bementioned Aspergillus nidulans, Aspergillus niger, Aspergillusochraceus, Aspergillus gigantius,

Aspergillus amsterlodami and other Aspergillus species, Rhizopnsnigricans, Rhizopus arrhizus and other Rhizopus species, Neurosporasitophila and other Neurospora species, Pestalotia foedans and otherPestalotia species, Dactylicum dendroides and other Dactylicurn species,Cephalothecium roseum and other Cephalothecium species, Absidia glaucaand other Absidia species, Delacroixia caromzta and other Delacroixiaspecies, Tricothecium roseum and other Tricothecium species, Thammdiumelegans and other Thamnidium species, Syncephalostrum racemonsum andother Syncephalostrum species, and certain Penicillium species.

In general, the conditions of culturing the microorganisms for thepurposes of this invention are (except for the inclusion of the steroidto be converted) the same as those of culturing microorganisms for theproduction of organic acids, antibiotics, etc., i.e., the microorganismis aerobically grown in contact with (in or on) a suitable fermentationmedium; A suitable medium essentially comprises a source of nitrogenousfactors and a source of carbon and energy. The latter may becarbohydrate (such as sucrose, molasses, glucose, maltose, starch ordextrin), a higher fatty acid, a fat and/or the steroid itself.Preferably, however, the medium includes an assimilable source of carbonand energy inaddition to the steroid. The sources of nitrogeneousfactors may be organic (e.g., soybean meal, corn steep liquor, meatextract and/ or distillers solubles) or synthetic (e.g., composed ofsimple, synthesizable organic and inorganic compounds such as ammoniumsalts, alkali nitrates, amino acids or urea).

An adequate sterile-air supply should be maintained during fermentation,for example, by the conventional methods of exposing a large surface ofthe medium to air or by utilizing submerged aerated culture. The steroidmay be added to the culture during the incubation period or included inthe medium prior to sterilization or inoculation.

Suitable starting steroids include theoa-haloproge's terones (e.g.6a-fluoroprogesterone and 6a-chloroprogesterone), the6u-halo-l-dehydroprogesterones (e.g. 6ozfluoro-I-dehydroprogesterone),the 6u-halo-17-u-hydroxyprogesterones (e.g. Ga-flnoro-lMhydroxyprogesterone),

however, since the Zl-ester is hydrolyzed during the microbialconversion these esters are in effect equivalent to the free 21-hydroxylcompounds. In "those cases where the l-dehydro starting steroids are newcompounds,

they can be prepared from the corresponding 1,2-saturated derivatives bymicrobial dehydrogenation, using for example Bacterium cyclo-oxydans andthe procedure disclosed in US. Patent No. 2,822,318.

r If the lla-hydroxy steroids formed contain a 2l-hydroxyl group (Y ishydroxy), the compound is then esterified in the 21-position to protectthis group. This esterification is accomplished in the usual manner bytreating the steroid with one mole equivalent of an acylating agent suchas an acyl halide or acid anhydride in the presence of a basic agent.Suitable acylating agents include the acyl chlorides and acid anhydridesof hydro carbon carboxylic acids of less than ten carbon atoms, asexemplified by the lower alkanoic acids (e.g. acetic, propionic andhexanoic acid), aromatic carboxylic acids (e.g. benzoic and .toluicacid), aralkanoic acids (e.g. phenacetic and B-phenylpropionic acid),cycloalkanecarboxylic acids, cycloalkenecarboxylic acids, and loweralkenoic acids.

The steroids thus formed having the general formula wherein the1,2-position is saturated or double-bonded, X and Z are as hereinbeforedefined, and Y" is hydrogen or acyloxy, are then converted to anlla-sulfonate by treatment with a sulfonating agent such as an organicsulfonyl halide, the reaction preferably being conducted in the presenceof a tertiary base, such as pyridine. Particularly preferred among thesulfonating agents are the lower alkane sulfonyl chlorides asexemplified by methanesulfonyl chloride and ethanesulfonyl chloride. Thereaction results in the preparation of the new lla-sulfonic acid estersof this invention having the general formula CHzY (i= ---z RS020" /1\EXAMPLE 1 6 m-fluoro-I 1 -epihy drocortison e [6 a-flu0ro-A pregn en e-11 (1,17u,21-tri0l-3,20-dione] (a) Fermentatian.-A medium of thefollowing composition is prepared:

G. Corn steep liquor 36 Brown sugar NaNO 6 CaCO s Lard oil 2 KH PQ, 1.5MgSO -7H O 0.5 ZnSO (1% solution) 0.1

Distilled water to make one liter. Steam-sterilized for 30 minutes atpsi.

The .pH of the medium is adjusted to 7.0:04 (with sodium hydroxidesolution); and 50 ml. portions of the medium are distributed in 250 ml.Erlenmeyer flasks and the flasks plugged with cotton and sterilized byautoclaving for 30 minutes at 120. When cool, each of the flasks isinoculated with one ml. portions of a vegetative inoculum of Aspergillusnidulans (ATCC 11267), prepared as described hereinafter. The flasks aremechanically shaken for 22 hours on a rotary shaker (280 cycles/ min.-2inch radius) in a room maintained at 25, after which time 10% transfers(by volume) are made to 250 ml. Erlenmeyer flasks (sterilized asdescribed above), each containing 50 ml. of the following medium:

Prior to inoculation, to each of the second series of flasks is added0.25 ml. of a 60 mg./ml. solution of Got-fluoro- Compound S (6a-fluoro Apregnene-l7a,2l-diol-3,20- dione) in N,N-dimethylforrnamide. Theseflasks are then incubated under the same conditions as used for thefirst series of flasks for l00 hours, after which the contents of theflasks are pooled and filtered by suction through Seitz clarifying pads.[The vegetative inoculum used is grown from stock cultures (lypohilizedvial or agar slant) for three weeks in a medium of the followingcomposition: glucose, 20 g.; yeast extract, 2.5 g.; K HPO l g.; agar, 20g.; and distilled water to make one liter. For inoculation the surfacegrowth was suspended in 5 ml. of a 0.01% Dupanol solution] (b) Isolationof 6c: fluoro 11 ep hydrocortisone. 4,940 ml. of the culture filtrateobtained in a (based on the fermentation of 1.41 g. of6a-fluoro-Compound S) is extracted with four one liter portions ofmethylisobutyl ketone and the combined extracts evaporated to dryness invacuo. The crude residue (about 970 mg.) is dissolved in a small amountof chloroform and after cooling the crystals are separated byfiltration. Stepwise concentration of the chloroform mother liquorsyield 3 sucoessive crops of crystals, all of which are recrystallizedfrom acetone-hexane to yield a total of about 520 mg. of crude6oc-fll10IO-1 l-epihydrocortisone. This on recrystallization fromacetone-hexane yields about 470 mg. of the pure compound having thefollowing properties: M.P. about 201-203, [a] +100 (c, 0.46 in ethanol);

a533,, 236 m (e=14,000); my 2.88, 2.97, 5.86, 6.04, 6.2011

Analysis.-Calcd. for C H O F(380.44): C, 66.26; H, 7.67; F, 5.11. Found:C, 66.37; H, 7.77; F, 4.78.

From the combined acetone-hexane mother liquors is obtained a secondisomeric substance, 6a-fluoro-A -pregnene-lSfi,l7a,21-triol-3,20-dioneof the following properties: M.P. about 225-228; [a] +79 (c, 0.34 in 95%ethanol); M21 236 m (e=15,900); A313 2.97, 5.85, 6.05 and 6.18

Analysis-Calcd. for C H O 'F(380.44); C, 66.26; H, 7.67. Found: C,66.02; H, 7.66.

When Aspergillus niger (Wis 72-7) or Rhizopus nigricans (Rutgers-86) issubstituted for the Asperigillus nidulans in Example 1, the same6a-fluoro-11-epihydrocortisone product is obtained.

EXAMPLE 2 6u-chloro-1I-epihydrocortisone Following the procedure ofExample 1, but substituting the same amount of 6a-chloro-Compond S forthe 60:- fiu0ro-Cornpound S, 6u-chl0ro-11-epihydrocortisone is obtained.1

5 I M I EXAMPLES 6a-fluo'ro 11 04,17a-dihydroxyprogreslerone Followingthe procedure of Example 1, but substituting an equivalent amount of6a-fluoro 17u-hydroxy. progesterone for the 6a-fluoro-Compound S,6a-fluoro-1'1a,17adihydroxyprogesterone is obtained.

EXAMPLE 4 6a-fluor0-A -pregnad iene-I 1 ea] 7a,21-tri0l-3,20 -dineEXAMPLE 6'a-flu0r0-11-epihydrocorii$one 21 -acetate To a solution of 239mg. of a-fluoro-ll-epihydrocortisone in 1 ml. of dry pyridine is added1.65 ml. of a solution of 216 mg. of acetic anhydride and 5 ml. ofpyridine. The mixture is allowed to stand at room tein: perature for 3%hours, after which the reagents are evaporated in vacuo. The residue istaken up in ethyl acetate and the resulting solution evaporated again invacuo in order to remove traces of acetic acid and pyridine. Theresulting residue of 6a-fluoro-ll-epihydrocortisone 21-acetate is usedwithout further purification in the mesylation reaction of Example 7.

for the methanesulfonyl chloride in Example 7 the correspondingll-ethanesfulonate is obtained.

EXAMPLE 8 6oc-flu0ro-A -pregnadiene-11a,1 7a,21-triol+3,20- dione-I 1a-mesylate 21 -acetate Following the procedure of Example 7 butsubstituting 6a-fluoro-A -pregnadiene-11a,17a,21-triol-3,20-dione 2 1-acetate for the 6u-fluoro-1l-epihydrocortisone ZI-acetate, there isobtained 6a-fiuoro-A -pregnadiene-1la,17a,21- triol-3,20-dionella-mesylate ZI-acetate.

Similarly, 6a-chloro-1l-epihydrocortisone ZI-acetate, 6a-fluoro-A-pregnadiene-11u,2l-diol-3,20-dione 2l-acetate,6a-fluoro-1l-epicorticosterone 2l-acetate, 6u-fluoro-1let-hydroxyprogesterone, 60c flHOI'O A pregnadiene- 1la,17a-diol-3,20-dione,' 6ix-fiuoro-l lu,l7a-dihydroxyproprogesteroneand 6a-fluoro-n -pregnadiene Ila-016,20

dione yield their corresponding lla-mesylate derivatives. EXAMPLE 9 Asolution of 232 mg. of '6o t-fluoro-ll-epihydrooortisone ZI-acetatell-mesylate and 520 mg. of anhydrous sodiumacetate in 5 m1. of glacialacetic acid is refluxed for /2 hour. The mixture is diluted with Water,extracted with chloroform, and the chloroform extract Washed withbicarbonate until neutral and finally with water. The

" extract is dried over sodium sulfate and the solvents re- Similarly,if another acylating agent is used instead of acetic anhydride, thecorresponding 21-ester if formed. Thus, propionic anhydride yields theZI-propionate and benzoyl chloride gives the 21-benzoate.

EXAMPLE 6 6 a-flu0ro-A -pregnad iene-I 1 a,] 7 a,21 -triol3,20- dione 21-acetate Following the procedure of Example 5, but substituting 238 mg.of 6ot-fiuoro-A -pregnadiene-llu,l7a,21- triol-3,20-dione for the6a-fluoro-1l-epihydrocortisone, 6u-fluoro-A -pregnadiene-1 1a, 17a,2l-triol-3,20-dione 21- acetate is obtained.

Similarly, 6ot-chloro-ll-epihydrocortisone, 6a-fluoro- Apregnadiene-1la,2l-diol-3,20-dione, and 6e-fluorol lepicorticosteronecan be converted to their respective 21- acetate derivatives.

EXAMPLE 7 6 ixuoro-l 1 -epihydr0c0rtisone 21 -acelate 1 1 -mesylate To asolution of 270 mg. of the dried residue obtained in Example 5 in 3 ml.of dried pyridine is added at 0 .15 ml. of methanesulfonyl chloride. Thereaction is allowed to proceed for 18 hours, after which time excessmethanesulfonyl chloride is destroyed by the addition of a small pieceof ice. Water is then added and the mixture extracted with chloroform.The chloroform extract is washed with dilute hydrochloric acid, dilutesodium bicarbonate and finally with water, dried over sodium sulfate andthe solvent removed in vacuo. The mesylate acetate crystallizes readilyfrom acetone-hexane, the pure substances having the followingproperties: M.P. about 140-142" (dec.); 1x1 9 4404 (c, .59 inchloroform); M2,, 243 m (e=l4,900); A235,? 2.87, 3.00, 5.72, 5.79,

5.94, 6.18, 7.53 and 8.6

Analysis.--Calc. for C H O SF-H O: C, 55.64; H, 6.80. Found: C, 56.01;H, 7.00.

If 0.15 ml. of methanesulfonyl chloride is substituted moved in vacuo.The residual 9,ll-dehydro compound after crystallization fromacetone-hexane has the following properties: M.;P..220-221; [a] +64 (c,.40 in acetone). Chromotog'rap-hy ofthe mother liquors on acid washedalumnia gives an additional amount of the above compound in elution withbenzene. Elution with 10% of chloroform in benzene gives a small amountof the 11,2l-diacetate of 6oz-fiuoro epi F of the following properties:M.P. about 215-217; [a] (c, 0.41 in CHOl I MR}, 243 mu (6=16,9OO)6; A5312.90, 5.77, 5.82, 5.96,

Analysis.-Ca1c. for C H O F(464.5): C, 64.65; H, 7.11. Found: C, 64.85;H, 7.17.

EXAMPLE 10 6 ccaura-A -pregnatriene-17a,2I-di0l-3,20- dione 21 -acetateFollowing the procedure of Example 9, but substituting 230 mg. of6a-flu0ro-A -pregnadiene-1la,17a,21-triolepihdrocortisone 21 acetate 11mesylate, 6a. fluoroepihydrocortisone 21 acetate 11 mesylate, 6oz[fluoro- A -pregnatriene-l7a,2'l-diol-3,20-dione ZI-acetate is obtained.

Similarly, by following the procedure of Example 9, 6a-chloro 11epihydrocortisone ll-mesylate ZI-acetate, 60o fluoro A-pregnadiene-l1a,2l-diol-3,20-dione 1-1- mesylate 2lacetate,6a-fluoro-1l-epicorticosterone 11- mesyiate ZI-acetate,6OL-fillOIO-1IOL-hYdIOXYPI'OgCSteI'OHC 1 l-mesylate, 6wfiuoro-A-pregnadiene-l 1u-ol-3,20-dione ll-mesylate,6a-fiuoro-11a,l7a-dihydroxyprogesterone l1- mesylate, and 6a-fil101'0 Apregnadiene-l 10,17a-di0l- 3,2'0-dione ll-mesylate, yield 6ot-chloro-A-pregnadiene :,21 diol 3,20 dione 2l-acetate, 6a-fluoro- A-pregnatriene-21 o1 3,20 dione 2l-acetate, 6afiuoro-A-pregnadiene-21-ol-3,20-dione 2l-acetate, 6afluoro-A-pregnadiene-3,ZO-dione, 6ot-fluoro-A pregnatriene 3,20 dione,6u-fluoro-A -pregnadiene- 17a-ol-3,20-dione, and 6a fluoro M-pregnatriene- 17 a-ol-3,20-dione, respectively.

The invention may be variously otherwise embodied within the scope ofthe appended claims.

wherein the nuclear carbon atoms in the 1 and 2 positions are connectedby a linkage selected from the group consisting of a single bond and adouble-bond, X is a halogen selected from the group consisting of chloroand fluoro, and Y is selected from the group consisting of hydrogen,hydroxy and the acyloxy radical of a hydrocarbon carboxylic acid of lessthan ten carbon atoms.

2. 6u-fluoro-1l-epihydrocortisone.

3. 6a-chloro-l l-epihydrocortisone.

4. 6oz. fluoro A -pregnadiene-11a,17a,2l-triol-3,2O- dione.

5. 6a-halo 11 epihydrocortisone 21-lower alkanoate, wherein the haloradical is selected from the group consisting of chloro and fluoro.

6. 6a-fluoro-1l-epihydrocortisone ZI-acet-ate.

7. A steroid of the formula wherein the nuclear carbon atoms in the 1and 2 positions are connected by a linkage selected from the groupconsisting of a single bond and a double-bond, X is a halogen selectedfrom the group consisting of chloro and fluoro, Y" is selected from thegroup consisting of hydrogen and the acyloxy radical of a hydrocarboncarboxylic acid of less than ten carbon atoms, Z is selected from thegroup consisting of hydrogen and hydroxy, and R is lower alkyl.

8. Get-halo 11 epihydrocortisone 21-lower alk anoate Ila-loweralkanesulfonate, wherein the halo radical is selected from the groupconsisting of chloro and fluoro.

9. 6m fluoro 11 epihydrocortisone 21-acetate 11amesylate.

References Cited in the file of this patent UNITED STATES PATENTS2,649,402 Murray et al. Aug. 18, 1953 2,819,264 Gould et al. Jan. 7,1958 2,838,498 Magerlein et al. June 10, 1958 2,838,499 Spero et al.June 10, 1958 2,838,501 Campbell et al June 10, 1958 2,852,511 FriedSept. 16, 1958 2,880,205 Campbell et al. Mar. 31, 1959 OTHER REFERENCESRingold et al.: I. Am. Chem. Soc., vol. 80, No. 23, page 6464 (1958).

1. A STEROID OF THE FORMULA